The alpha-crystallins comprise a large fraction of the soluble protein in the vertebrate lens where they were, for many years, believed to function solely as structural proteins. This small family of crystallins is encoded by only 2 genes, the alphaA- and alphaB-crystallin genes and is collectively referred to as alpha-crystallin. They are related to the small heat shock proteins, and in vitro they exhibit molecular chaperon activity, autokinase activity, single stranded DNA binding activity, and interact with and affect the state of several cytoskeletal components. alpha-Crystallin, especially alphaB-crystallin, has been shown to be a normal constituent of many non-lenticular tissues, and has been detected in cytoplasmic inclusion bodies found in several human pathological conditions. Toward understanding the major roles of alpha-crystallin in vivo, we are functionally deleting alpha-crystallin proteins by disrupting or knocking-out their genes in mice. We are attempting to elucidate the in vivo functions of alphaA- and alphaB-crystallin 1) in lens development and morphogenesis; 2) in maintaining a stable, transparent lens throughout the life of an organism (i.e. preventing cataract); 3) in the non-lenticular tissues where they are normally present; and 4) in non-lenticular pathological conditions. We have generated mice which lack alphaA-crystallin, mice which lack alphaB-crystallin, and mice which are deficient in both alphaA- and alphaB-crystallin. Our research has demonstrated that neither alphaA-crystallin nor alphaB-crystallin is essential for survival of the laboratory mouse. Mice deficient in either or both of the alpha-crystallins grow and reproduce normally. Since alphaB is highly expressed during embryogenesis in the developing heart and other structures, and is a normal constituent of adult heart, skeletal muscle and several other organs, it was thought to be an essential protein. Analysis of alphaA-/- mice reveals that lens development occurs relatively normally, and although the wet weight of alphaA-/- lenses are approximately 35 % less than that of controls, the gross architecture of the lens is normal, exhibiting an anterior epithelial layer and elongated fiber cells. Cataract begins to form at 7 weeks of age or earlier and eventually the lens becomes completely opaque. Dense proteinaceous cytoplasmic inclusion bodies (1 to 3 mu in diameter) form in the fiber cells of these lenses as early as 2 to 4 weeks of age. These spheroidal bodies have been isolated and found to be almost entirely alphaB-crystallin, and modified forms of this protein. Mass spectroscopic analysis has identified mono- and di-phosphorylated forms of alphaB-crystallin, and un- mono- and di-phosphorylated forms of a truncated alphaB-crystallin polypeptide as constituents of these inclusion bodies. As the relative amount of alphaA-crystallin decreases, the phosphorylation of alphaB-crystallin increases. This is consistent with an observation that human Down's syndrome patients, carrying 3 copies of the alphaA-crystallin gene, exhibit decreased phosphorylation of alphaB-crystallin in the lens. Mice lacking alphaB-crystallin exhibit no apparent phenotype when maintained under animal facility conditions. The lenses and other tissues in which alphaB-crystallin is normally expressed do not appear to be affected by the absence of alphaB-crystallin in young mice and young adults. We are currently studying older mice (8 to 12 months old) to determine how the absence of this chaperone/small heat shock protein might affect the aging process. Mice lacking both alphaA- and alphaB-crystallin are microphthalmic and have severe cataract characterized by disorganized fiber cells. The newly differentiated lens fiber cells fail to migrate along the posterior lens capsule, and instead migrate only aneriorly, forcing the lens nucleus to the posterior of the lens. The lens fiber cells in these mice do not contain the inclusion bodies seen in alphaA-crystallin knockout mice, a finding consistent with alphaB-crystallin being the major component of those inclusion bodies. Other cellular events occurring during formation of these lenses are currently under study.